【作者】 陈开健；

【导师】 章怀云；

【作者基本信息】 湖南农业大学 ， 基础兽医学， 2003， 硕士

【摘要】 草鱼年产量占我国淡水鱼总产量的20％。其生长快、饲料来源广、饲养成本低和肉味鲜美。但草鱼病害多，危害严重，目前草鱼由苗种养至成鱼的成活率仅为10-15％，每年给我国淡水养殖业造成数亿元经济损失。特别是病毒性的草鱼出血病，流行季节长，发病死亡率高，因此，对草鱼出血病的有效防治一直是世界各国水产科学工作者艰苦探索而又未能找到理想解决办法的世界性难题。现代分子生物学技术突飞猛进的发展为我们切底解决这一难题提供了可能性和有效途径。综观国内外近些年来对转基因鱼的研究及结果证明，通过转基因定向培育出符合人们所需要的优良鱼种是完全可能的，也是十分有意义的。通过基因转移进行鱼类抗病育种研究，是鱼类转基因研究中的一个新的方向。 人-α干扰素(hu-α-IFN)是人体因病毒感染或其它诱生剂作用等所产生的一类非特异性抗病毒物质，是调节细胞功能的一类重要的糖蛋白。其作用是通过与细胞膜上的特异性受体结合，引发级联性的信号放大过程，将信号最后传递到细胞核内，对一系列基因的表达进行调控，并引发各种生理反应，整体性地提高自身的免疫和抗病毒能力。本研究的目的在于以人的α-干扰素基因(IFN-α)作为目的基因，与鲤鱼β-肌动蛋白基因启动子在体外重组，利用原核显微注射转基因技术将人-α干扰素基因导入草鱼基因组而开展的抗病转基因草鱼育种研究，其结合了干扰素和基因工程育种抗草鱼出血病病毒的优点，以期获得对草鱼出血病具有天然抗性的转基因鱼，并在此基础上培育出草鱼抗病新品系。从而探索出一种从根本上解决草鱼出血病的有效途径。 实验通过将hu-α-IFN基因重组分子采用显微注射法将这种重组基因转化草鱼1-2细胞期的受精卵，即以Narashige显微注射仪，将5nl DNA(约10~6～10~7分子)直接注射到选择好的受精卵核的动物极上。2000年共显微注射32197粒草鱼受精卵，出苗12945尾草鱼鱼苗，经孵化培育最后获得1120尾五寸左右草鱼，对1120尾草鱼提取血液总DNA进行PCR和Southern杂交检测，2000年转导的草鱼的PCR检测结果，有672尾草鱼即600k转化个体的总DNA能扩增出一条485kb的特异分子带，说明转化个体整合有huJ－IFN基因，经Southern杂交进一步证实，转入的抗病相关基因己在转基因草鱼体内染色体上得以整合，但整合位点没有固定区域，整合的拷贝数存在较大差异。对 PCR呈阳性的草鱼 672尾个体进行 ELISA检测，在 158尾草鱼中检出有人a－干扰素的表达即阳性率为23．5％，但基因表达水平在个体间有很大差异，对ELISA检测呈阳性的转基因草鱼接种草鱼出血病病毒的攻毒试验，证明了转基因鱼对草鱼出血病毒的抗性比普通草鱼提高了60｛0儿更多还原

【Abstract】 The annual yield of grass carp covers 20% in total output of the fresh water fish,which is fast growing and tastes delicious,besides, the forages of grass carp are various ,with a lower cost of raising,However, at present,the serious diseaes of grass carp results in a lower survival rate 10-15%,which cause more then billions yuan economic losses per year to the fresh water cultiuation ,especially, Hemorrhagic virus of carp lasts for a long period of time and features a high death rate,therefore,it’ s a woed-wide problem for the aquacultural scientific researchers to discover an effective settlement ,which is still under the discussion. The rapid development of modem molecular biology technique suppies a possible and efficient way for us to solove the problem completely with a comprehensive researches results to transgenic fish in recent years both domestic and aboard,it proves that it’ s possible and makes great senses to cultivate a high quality fish to meet the satisifaction of the people . Making anti-disease breeding researches of fish by transfer gene is a new direction in transfergene researches offish.Hu-α -IFN is a sort of biological activity mass of broad-spectrum antivirus and have evdient effictiveness to varities of viral-disease . it was important glucoprotein modulating cell functions by combination to idiosyncrasy receptor on cell membrane, which inducing process of cascade signal magnifying, then passing signals into nucleus to induce the modulation of the serial gene expression and viarous physiological reaction, and inhence immunity and antivirus. Take advantage of the character of hu- α -IFN, The recombination gene was recombinated hu- α -IFN gene and carp β -actin gene promoter, then it was migrate into the body of lower vertebrate grass carp. Through studing its integration, expression and antivirus, expected to gain a new kind of grass carp assumed notable antivirus of Hemorrhagic virus of carp . the research adopts that Hu- α -IFN gene were introduced into the nuclei of oocytes or cytoplasm of Grass carp to develop anti-disease transgenic grass carp breeding researches,combing the advantage of Hu- α -IFN gene and breeding by Genetic engineering,with an aim of finding out an effective way of solving antivirus of Hemorrhagic virus of carp completely.IN research of transgenic fish , Hu- α -IFN gene ( recombination gene) is cutdown and introduced into the nuclei of oocytes or cytoplasm of Grass carp at one-cell or two-cell stage via micyoinjection by Narashige micyoinjection apparatus . the recombination gene micyoinjected is 5nl DNA (about 106-107) into everyone oocytes or cytoplasm of Grass carp. There were 32197 oocytes of 12 series via micyoinjection and achived 12945 fries in 2000 year , By hatching and raising .Genomic DNA was extracted from Grass carp who were transformated in 1120 Grass carp presented postive reaction by blood . The transgenic Grass carp were detected by PCR , Southern blot. The results showed that PCR applify a 485 specific molecular band in 672 individuals, the rate of positive reaction is 60%, Southern blot result shows a strong signal in transgenic fish . It is concluded that Hu- α -IFN gene has been integrated and expression . It is also that foreign gene integrating position and copy number is different individuals , By ELISA detecting , 23.5% transgenic fish’ were detected Hu- α -IFN gene expression in all 672 transgenic fish , but Expression level of Hu-α -DFN is significant difference in different individuals and growth period.it is also conformed that transgenic Grass carp presented masculine in ELISA detecting have more resistivity than control Grass carp in inoculatived GCHV testing , the rate is 60-70% .更多还原